Significance HIV is primarily transmitted by sexual contact. Thus a vaccine must be able to prevent sexual transmission of HIV. In order to test candidate vaccines in the SIV/rhesus macaque animal model, SIV stocks must be titered by vaginal inoculation in order to identify the minimum inoculum capable of infecting 100% of animals inoculated. Objectives The present study was designed to titer a SIVmac251 stock by vaginal inoculation of rhesus macaques. Results Two rhesus macaques were inoculated intravaginally with 105 50% tissue culture infectious doses, both became infected. Six rhesus macaques were inoculated intravaginally with 104 50% tissue culture infectious doses, 3 became infected. Six rhesus macaques were inoculated intravaginally with 103 50% tissue culture infectious doses, 2 became infected. Six rhesus macaques were inoculated intravaginally with 103 50% tissue culture infectious doses, 1 became infected. Future Directions We have identified a dose of SIVmac251 which transmits by vaginal inoculation to all inoculated rhesus macaques. This virus can be used to vaginally challenge animals immunized with candidate HIV vaccines. KEYWORDS viral pathogenesis, SIV vaginal transmission, vaccine challenge model